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WO2006049336A1 - Méthode de préparation de cellules souches et médicament réparateur de tissus - Google Patents

Méthode de préparation de cellules souches et médicament réparateur de tissus Download PDF

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Publication number
WO2006049336A1
WO2006049336A1 PCT/JP2005/020755 JP2005020755W WO2006049336A1 WO 2006049336 A1 WO2006049336 A1 WO 2006049336A1 JP 2005020755 W JP2005020755 W JP 2005020755W WO 2006049336 A1 WO2006049336 A1 WO 2006049336A1
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Prior art keywords
cells
stem cells
liver
differentiation
cell
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PCT/JP2005/020755
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English (en)
Japanese (ja)
Inventor
Hidechika Okada
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OKADA ALAN
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OKADA ALAN
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/407—Liver; Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/14—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from hepatocytes

Definitions

  • the present invention relates to a method for preparing a stem cell for pluripotency, to prepare a cell, and to a table including a cell in which a stem cell is induced to differentiate.
  • Patent Document 1 is mainly composed of dendritic fibers having an antigen-presenting cell ability obtained by culturing under a cell sap differentiation inducing agent containing such ⁇ -dendritic fiber precursor cells and chemokines. Textile vaccine. "It is shown.
  • Patent Document 2 describes a culture medium having a living body that cultivates human stem cells containing ⁇ human and a ligand protein as active ingredients and maintains or amplifies the number or frequency of human stem cells. And “a culture method in which human stem cells and human cells using this culture medium are cultured under conditions that allow reversal of the ligand protein, and the number or frequency of human stem fibers is increased or amplified.”
  • Human CNS neural stem cells contain mitotic iffil factors (such as epithelial cell BS ⁇ growth factors, basic glioblast growth factors, etc.), as well as their rodent homologues. When maintained in a blue-free culture medium, it grows in suspension culture and forms what is known as the “neosphere.
  • Patent Document 3 discloses the isolation of this human central nervous system stem fiber and the method and medium for growing, differentiating and f-planting these cells. Has been.
  • Patent text For example, for this month : «, a composition of mammalian liver transplant cells isolated from cocoons, wherein at least 80% of the cocoons in the stringer are R2.
  • Patent Document 1 JP 2000-143534 A
  • Patent Document 2 WO 02/16556 A1
  • Patent Literature 3 Special Table 2003— 512333
  • Patent Literature 4 Special Table 2004—531270 ⁇ Report Invention Disclosure
  • the present invention it is not necessary to administer a drug such as an immunosuppressive agent that does not worry about an immune reaction even after transplanting the stem cells prepared using its own moon cake.
  • a drug such as an immunosuppressive agent that does not worry about an immune reaction even after transplanting the stem cells prepared using its own moon cake.
  • a method for obtaining a large amount of such pluripotent stem cells from the liver will be shared.
  • the purpose of this study is to improve the ability of the stem cells with multipotency in the liver to induce differentiation into various fibers including god follicles, thereby facilitating the transplantation of the fibers.
  • EmbrioniG Stem-cell canine stem fibers are obtained.
  • Stem cells can be removed from the cells and induced to differentiate into cells of the nervous system and other necessary tissue fibers. is there. In this case, it is difficult that there is no fear of refusal reaction because it uses its own stem fiber.
  • pluripotent stem cells are trapped in dust, instead of generating a regenerative moon, a part of the liver cirrhosis is taken out with / i; t ⁇ , ⁇ , etc.
  • Stem cells with abilities can be induced to differentiate into the target cells in vitro, and ⁇ or tissue binding C can be used for transplantation.
  • stem cells are extracted from the regenerated liver from the Ml of a mouse that has been introduced with a C * en Fluorescent Protein (GFP) gene, which has a fluorescent property, so that all cells emit fluorescent light. It can also be shown to be induced to differentiate into various organs by transplanting into a skid mouse.
  • GFP en Fluorescent Protein
  • Moon Tfli has a pluripotent stem that can be used for transplantation treatment by inducing differentiation into various cell professions including god follicles.
  • stem cells created from the patient's own ffl are used, immunity will not occur during transplantation, so there is no need to administer drugs such as immunosuppressants.
  • it has pluripotency! ⁇
  • To create a large number of follicles from the liver cut part of the patient's liver! It is possible to use regenerated liver fibers that proliferate quickly. Simple drawing
  • Fig. 1a shows a 1-week Sphere (fine BS ⁇ demon) created from fetal liver on day 13 of embryonic day.
  • Figure 1c shows that 10% of the cells stained positive with Nest.
  • Fig. 2a shows that when the spheres formed in one week are induced to differentiate into gods, cells migrate from the spheres and spread on the seventh day.
  • Figure 2b shows that the cells that migrated are single.
  • Figure 2c shows a two-part configuration
  • Figure 2d shows the divergence and the form of God.
  • Fig. 3a shows the cyst-like morphology on the 7th day of induction of differentiation into gods ⁇ , and the specific staining characteristic of the god cells stained with the fluorescent antibody against Tubulin was observed.
  • FIG. 3b shows that difficult specific staining was observed in the god cells that stained with a fluorescent antibody against MAP-2.
  • Fig. 3c shows that the characteristic staining characteristic of the vesicles stained with the fluorescent antibody of NeurofilamentrH was observed.
  • Fig. 3d shows that leaky specific staining was observed in the mycelium that stained with the fluorescent antibody against GAD.
  • the counter dyeing is shown with Hoechs 33258 dyed in force ⁇ .
  • Figure 4a shows cells cultured for 14 days in fetal liver-derived sputum in moon cake medium.
  • Fig. 4b is a photograph of the electrode glass of a fiber 1 clamp cultured for 14 days in a cell for inducing differentiation into mesenchymal cells.
  • Fig. 4c shows the voltage ggted polasium current, which is the differentiated cell of the vesicle-like I.
  • Figure 4d shows that the response of Figure 4c is ifllfljed with channel blockers ⁇ 6.
  • FIG. 4g shows that cells grown in stem cell culture medium were not reactive with GABA.
  • Figure 4h shows that GABA senses the stimulus ⁇ .
  • Figure 4j shows that ⁇ lj is perceived at Seroto ⁇ .
  • Fig. 5a shows fetal fermentation on day 13 of embryogenesis: U-sections are fluorescently stained with an antibody against albumin and a large number of fibers are shown to be 11 ⁇ .
  • Figure 5b shows that when IH is fluorescently stained with an antibody against estin in green, Nesuth-positive ⁇ is interspersed between stem cells.
  • Figure 5c shows that in the interstitial strips of adult mice, most of the cells stained by Nesun can be detected.
  • Figure 5d shows that adult mice grow as a result of excision of about two-thirds of the moon cake. On the third day of live liver thread, Nestin positive 1 is observed as many as fetal liver. ing. BEST MODE FOR CARRYING OUT THE INVENTION
  • stem cell as used in the present invention is a cell derived from a fetus or an adult tissue and is defined as a cell that can be differentiated into a plurality of cell lineages, and includes human neural stem cells, human mesenchymal stem cells, human 5 ⁇ 1 stem «, human lunar Tfl stem cells, etc. are included.
  • “Human stem « ” is a cell derived from fetus or adult silkworm, and is defined as a cyst that can differentiate into any night cells] ⁇ The
  • the ⁇ cells were washed with 20n ⁇ mEGF (epidermal growth fector: Sgma), 40 ng / ml bEGF (epidermal growth -factor: Kuroko, 10 ul / ml N2 supplement (L itro ⁇ n 3 ⁇ 4: g3 ⁇ 4 v to 100 U / ml Cooperlin G, 100 ug ml streptomycin, 100 ng ml 7 f; H 1 lysine B ( The cells were suspended in a DMEM / F12 culture medium containing Invitrcgen's cells and cultured for 1 week in a culture coat coated with poly + EMA (poly 2-hyc) xyethy) methacrylate: Sigma. Many «were killed but some fibers began to grow and began to form spheres, cultivated twice a week. Further cultivating I while changing the nutrient solution I Sphere is thin until the second week.
  • neuiOfilamenHH (NF "H) (Fig. 3c) was stained positively, respectively, and showed the characteristics of adult cells.In contrast to Jd, the expression of GFAP or 04 antigen, which is a specific source of glial cells, was not observed. Therefore, it was shown that differentiation induction specialized for nerve cells is possible.
  • GAD known as a marker of GABA-reactive vesicles
  • GABA-reactive vesicles is not expressed in peripheral nerves
  • 62.4% of GAD positive fibers were observed, and the differentiation was induced in the central nervous system god follicles, as shown in Fig. 3d.
  • Example 1 In the method of Example 1, it was proved by a physiologic method that Ken, which was induced to differentiate from fetal liver to god cells, had the function of god fibroblasts. Differentiation induction On day 14 of culture, the electrical potential of the membrane was minus 26 +/- 9.7 mV. Therefore, as shown in Fig. 4a and Fig. 4b, the ion current was measured by the clamp method. Increasing 1E showed outward folds in 87% of cells. Since it was blocked by channel blockers ( ⁇ -CI and 4 ⁇ amynopyridne), as shown in Fig. 4c and Fig. 4d, the voltage-dependent "utrent function of the neurofilament was induced to differentiate into neuronal fibers. The ionic current was 1 micromole of Tetrodotoxin (TTX) added to the external solution, and even if it was not blocked, it was insensitive to 1 cell. In addition, the action: the potential was not accepted up to 10pA.
  • liver of embryonic day 13 mice was fluorescently stained in red with an antibody against albumin, as shown in Fig. 5a, most cells were sensitive to alb 5.
  • fluorescently stained Darin with this antibody against fetal ⁇ 3 ⁇ 4 Nesti'n as shown in Fig. 5b, the long protrusions appear and the cells are scattered between albumin-positive stem cells: It was.
  • Nesutin-positive cells were almost detected as shown in Fig. 5d;
  • ⁇ Cells were detected, and it was confirmed that the regenerating liver had many different ability to differentiate into the nervous system, like the fetal liver. Used as a source of stem cells Therefore, it is possible to differentiate into cells and transplant them to the patients themselves. It shows that medical care becomes a function.
  • GFP Green Fluorescent Protein
  • the stem cells in the regenerating liver have pluripotency and have similar characteristics to ES cells. Therefore, by inducing differentiation of stem cells from regenerated liver in the same way as ES fibers, cells for transplantation to patients themselves, transplantation fibers, organs for transplantation, or dislocation of patients to patients with neurodegenerative diseases It can be used for live medical care.
  • Glucose 02 ⁇ . ⁇ preferably 0.6% W / V
  • EGF Q2 ⁇ 200 g ml, girls or 20ng ml

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Developmental Biology & Embryology (AREA)
  • Neurology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Wood Science & Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Neurosurgery (AREA)
  • Urology & Nephrology (AREA)
  • Botany (AREA)
  • Physiology (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Nutrition Science (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)

Abstract

La présente invention a pour objet l’induction de la différentiation de cellules souches pluripotentes du foie en diverses cellules incluant des cellules nerveuses et des tissus cellulaires, et l’emploi desdites cellules différenciées dans le cadre d’une transplantation. À partir du foie d’un patient, un foie régénéré est obtenu et des cellules souches en sont extraites. Puis, on induit la différenciation des cellules souches en cellules nerveuses ou en cellules d’autres tissus, avant de transplanter lesdites cellules au sein des cellules ou tissus défectueux du patient. Grâce à ces cellules souches autologues, il est possible d'obtenir des cellules souches pluripotentes sans risque de rejet. Une fraction du tissu hépatique est prélevée, par exemple par biopsie, et la différenciation des cellules souches pluripotentes qu’il contient en cellules ou tissus cibles est induite in vitro. Ceci permet d'obtenir un médicament réparateur de tissus contenant les cellules souches à transplanter.
PCT/JP2005/020755 2004-11-05 2005-11-04 Méthode de préparation de cellules souches et médicament réparateur de tissus Ceased WO2006049336A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004-321870 2004-11-05
JP2004321870A JP2008029201A (ja) 2004-11-05 2004-11-05 幹細胞の調整方法と組織治療剤。

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WO2006049336A1 true WO2006049336A1 (fr) 2006-05-11

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JP6185907B2 (ja) * 2011-03-30 2017-08-23 セルラー ダイナミクス インターナショナル， インコーポレイテッド 神経分化のための多能性幹細胞の予備刺激

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064748A2 (fr) * 2001-02-14 2002-08-22 Furcht Leo T Cellules souches adultes totipotentes, sources de ces cellules, procedes d'obtention et de maintien de ces dernieres, procedes de differentiation de ces cellules, procedes d'utilisation correspondants et cellules derivees des cellules susmentionnees

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002064748A2 (fr) * 2001-02-14 2002-08-22 Furcht Leo T Cellules souches adultes totipotentes, sources de ces cellules, procedes d'obtention et de maintien de ces dernieres, procedes de differentiation de ces cellules, procedes d'utilisation correspondants et cellules derivees des cellules susmentionnees

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DENG J ET AL: "Neural trans-differentiation potential of hepatic oval cells in the neonatal mouse brain.", EXP NEUROL., vol. 182, 2003, pages 373 - 382, XP008049057 *
HAO H N ET AL: "Fetal human hematopoietic stem cells can differentiate sequentially into neural stem cells and then astrocytes in vitro.", J HEMATOTHER STEM CELL RES., vol. 12, 2003, pages 23 - 32, XP002996373 *
XIAO-YAN S ET AL: "Expression of nestin, an intermediate filament protein, in human fetal hepatic stem cells.", ACADEMIC JOURNAL OF THE FIRST MEDICAL COLLEGE OF PLA., vol. 24, February 2004 (2004-02-01), pages 207 - 209, XP002996374 *
YANG L ET AL: "In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells.", PROC NATL ACAD SCI U S A., vol. 99, 2002, pages 8078 - 8083, XP002961837 *

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